1. Technical Field
The present invention relates to a method for producing luciferin (luminescent substrate) which reacts with a marine ostracod crustacean luciferase, and a novel marine ostracod crustacean luciferin compound and establishment of a technology for producing the same.
The present invention also relates to a derivative having a different luminescence wavelength from that of natural marine ostracod crustacean luciferin, a derivative and a composition having a low background, and a method for measuring luminescence.
2. Background Art
Luminescent crustacea, marine ostracod crustacean and its related species have a secretory luminescent enzyme (luciferase) and a luminescent substrate (luciferin), and the luciferin of marine ostracod crustacean emits a blue light with a maximum luminescence wavelength of 460 nm by an oxidation reaction using the luciferase of marine ostracod crustacean as a catalyst.
Since the luciferase of marine ostracod crustacean is characterized by being secreted extracellularly, when a cloned cDNA is used as a reporter gene, a synthesized protein is secreted extracellularly in mammalian cells and yeast cells. Accordingly, a luminescence activity of marine ostracod crustacean luciferase can be measured without disrupting the cell, and thus, for example, a gene transcription activity in the mammalian cell can be measured extracellularly (Non-patent Literatures 1 to 2, Patent Documents 1 to 3). Marine ostracod crustacean luciferase has been also used as secretory luciferase for a reporter assay in the yeast cells (Patent Document 4).
In reports using this luminescent enzyme, an example in which the secretion of a protein from the cell was visualized by performing an image analysis of this secretory luminescent enzyme (Non-patent Literature 3) and an example in which a change of the transcription activity in living cells was continuously measured by using the mammalian cells in which a reporter gene of marine ostracod crustacean inserting a transcription activity region of a growth hormone gene had been introduced (Non-patent Literature 4) are available. An example in which processing of a peptide from a protein was quantified by a fusion body of marine ostracod crustacean luciferase and a fluorescent protein (Patent Document 5) is also available.
In the field of drug discovery, it is important to develop and search protein expression inhibitors and secretion inhibitors, and screenings have been performed using the change of the gene transcription activity of a target protein in the cell as an indicator. It is a role of the reporter gene (protein) to report the change of the gene transcription activity due to an effect of the inhibitor. As the reporter protein, it is required not only to report on/off of the gene but also to have properties, e.g., being capable of analyzing the change of inhibitor effects with time (high time resolution), the reporter protein by itself not having the inhibitory effect, or the reporter protein not disturbing intracellular functions (non-cytotoxicity). As the reporter gene accomplishing the high time resolution and having no cytotoxicity, the reporter protein produced in the cell is required to be rapidly secreted or metabolized.
Marine ostracod crustacean luciferase is secreted and the secreted luciferase can be used to rapidly measure the change of the transcription activity extracellularly. Thus, the range of its use is wide. Although marine ostracod crustacean luciferase is such a useful reporter enzyme, its practical application and general use have been given up. This is because there is a great problem that luciferin, a substrate of luciferase, is not sufficiently supplied.
Problematic issues of marine ostracod crustacean luciferin include difficulty of stable supply of luciferin, self-luminescence of marine ostracod crustacean luciferin with albumin protein, and its luminescence wavelength overlapped with other luminescence systems.
In all previously reported syntheses of marine ostracod crustacean luciferin, an intermediate is etioluciferin which is a precursor. The synthesis of etioluciferin minimally requires 7 steps (Non-patent Literatures 5 to 7). If a yield is poor in the final step from etioluciferin to marine ostracod crustacean luciferin, it is necessary to prepare a raw material in a large amount. Thus, a cost for producing luciferin is remarkably increased. According to a method described in the previous literature, the yield of optically active luciferin synthesized by a condensation reaction of 3-methyl-2-oxovaleric acid with etioluciferin is 2% in 3 steps (Non-patent Literature 5, FIG. 1). This way, the yield in the final step is extremely low. Thus, it is actually difficult to produce optically active marine ostracod crustacean luciferin by organic synthesis on a commercial basis.
Racemic luciferin (Non-patent Literature 7) exhibits only about a half activity of native luciferin and has a luminescence background of non-native luciferin not depending on luciferase. Thus it is an extremely important issue in bioassay to synthesize optically active luciferin.
Meanwhile, marine ostracod crustacean luciferin and Renilla luciferin have an imidazopyrazinone skeleton as a basic skeleton. Thus, their maximum luminescence wavelengths are close and around 460 to 480 nm although they are somewhat different due to their luciferase structures. Thus, it is difficult to simultaneously measure these two luminescence systems.
There is the example in which the processing the active peptide from the protein was quantified by the fusion body of marine ostracod crustacean luciferase and the fluorescent protein. Strokes shift between the luminescence maximum (460 nm) of marine ostracod crustacean luciferase and the fluorescence maximum (525 nm) of the fluorescent protein is small which was about 60 nm. Thus, it was observed that the luminescence of marine ostracod crustacean luciferase interfered with the fluorescence of the fluorescent protein. This light interference causes the high background in quantification of the processing of the peptide. Thus, it has been desired to develop marine ostracod crustacean luciferin analogs having the different maximum luminescence wavelength.
Furthermore, marine ostracod crustacean luciferin causes chemiluminescence by reacting with albumin in culture media, although a quantum yield is low. Since the self-luminescence does not depend on the amount of luciferase, it is observed as the background in an intracellular imaging of marine ostracod crustacean luciferase and the measurement of the transcription activity. It has been desired to develop marine ostracod crustacean luciferin analogs having the low background.
A marine ostracod crustacean luciferin derivative has been synthesized and a chemiluminescence reagent used for the quantification of super oxide anion has been invented. However, no derivative which becomes the substrate of marine ostracod crustacean luciferase has been developed.    Patent Document 1: WO90/01542    Patent Document 2: JP 1991-30678-A    Patent Document 3: JP 2004-187652-A    Patent Document 4: JP 2005-169768-A    Patent Document 5: PCT/JP03/15828    Patent Document 6: JP 1993-60697-A Publication    Patent Document 7: JP 1993-286976-A Publication    Non-patent Literature 1: Thompson, E. M., Nagata, S. & Tsuji, F. I. Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells. Gene 96, 257-62 (1990)    Non-patent Literature 2: Nakajima Y, Kobayashi K, Yamagishi K, Enomoto T and Ohmiya Y: cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca. Biosci. Biotechnol. Biochem. 68, 565-70, 2004    Non-patent Literature 3: Inouye, S., Ohmiya, Y., Toya, Y. & Tsuji, F. Imaging of luciferase secretion from transformed Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA 89, 9584-7 (1992)    Non-patent Literature 4: Tanahashi, Y., Ohmiya, Y., Honma, S., Katsuno, Y., Ohta, H., Nakamura, H., Honma, K. Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase. Anal Biochem. 289, 260-6 (2001)    Non-patent Literature 5: Kishi, Y.; Goto, T.; Inoue, S.; Sugiura, S.; Kishimoto, H. Tetrahedron Lett. 1966, 3445-3450    Non-patent Literature 6: Karpetsky, T. P.; White, E. H. J. Am. Chem. Soc. 1971, 93, 2333-2334    Non-patent Literature 7: Nakamura, H. Aizawa, M. Takeuchi, D. Murai, A. Shimomura O. Tetrahedron Lett. 2000, 41, 2185